This invention relates to a method of diagnosing mammalian pathology or target tissues and more particularly to a photoidentification method of diagnosing pathology or target tissue using an optical imaging material containing an imaging agent and at least one auxiliary chromophore. The imaging material preferentially localizes in pathology or target tissue, absorbs light and in some cases fluoresces or phosphoresces upon exposure to light. The primary purpose of the auxiliary chromophore is prevention of photodamage to healthy tissue by the agent.
Certain classes of molecules, including, for example, synthetic porphyrin derivatives, naturally occurring porphyrins and their derivatives, chlorophylls and their derivatives, purpurins, phthalocyanines, other cyclic tetrapyrroles, and fullerenes can act as imaging, detection and diagnostic agents for pathologies or target tissues including tumors, atherosclerotic and arthritic tissue, and diseased blood vessels. Administration of these agents to a human or other organism results in preferential localization of the agent in any of a variety of pathologies with respect to surrounding tissue. Irradiation of the organism with light of a given wavelength or wavelengths results in absorption of light by the agent. In some cases, the agent then emits light by fluorescence or phosphorescence. Light absorption or light emission produces contrast between the pathology or target tissue and the surrounding tissue, and the detection of this contrast allows pathology or target tissue imaging, detection or diagnosis. Alternatively, agents of this type can be used to enhance contrast or otherwise improve detection in magnetic resonance imaging of pathology or target tissues or can bear radioactive isotopes whose detection can be the basis of pathology or target tissue imaging, detection and diagnosis, or can serve as contrast agents for X-ray radiological or other techniques involving high-energy radiation.
Absorption of light by the agent results in production of excited states. These excited states are by definition of higher energy than the original unexcited ground state of the agent. The excess energy can result in deleterious interactions with the organism. For example, excited triplet states of the agent (or singlet or other excited states) can react directly with tissue or other components of the organism to cause damage to the organism. Triplet states and other states of high multiplicity can also cause the formation of excited states of oxygen, such as singlet oxygen, and other powerful oxidizing agents, superoxides, and other oxygen radicals. These excited states of oxygen and oxygen radicals are known to cause damage to biological membranes as well as other components of the organism. The agent in an excited state can also react with other molecules present to create other species that are harmful to the organism. Such damage is not limited to pathology or target tissue, as the agent does not localize exclusively in the pathology or target tissue. After administration some agent is found throughout the organism, including the skin. This propensity to cause damage to healthy tissue in the organism can limit the usefulness of the agent for pathology or target tissue imaging detection and diagnosis.
Carotenoid pigments, which are ubiquitous in photosynthetic membranes, are essential for the survival of green plants. Three facets of carotenoid function are recognized in photosynthetic membranes. First, carotenoids photoprotect by rapidly quenching chlorophyll triplet states which are formed in antenna systems or photosynthetic reaction centers. This triplet-triplet energy transfer prevents chlorophyll-photosensitized formation of highly destructive singlet oxygen which is injurious to the organism. In addition, carotenoids act as antennas by absorbing light in spectral regions where chlorophyll absorbs weakly and by delivering the resulting excitation to chlorophyll via a singlet-singlet energy transfer process. Finally, nearby carotenoids quench chlorophyll first excited singlet states. This quenching has been ascribed to energy transfer or electron transfer or some other process leading to internal conversion and is believed to play a role in the regulation of photosynthesis.
A number of porphyrin materials have been found to localize in pathologies and damage that tissue upon irradiation with light. Many of these, such as xe2x80x9chematoporphyrin derivativexe2x80x9d and related materials, are being investigated as photodynamic therapeutic agents. All of these agents suffer from the problem that they are also absorbed by healthy tissue, which is consequently harmed by light.
Various synthetic carotenoids designed to mimic carotenoid photo protection have been investigated. Synthetic carotenoporphyrins consisting of a carotenoid part covalently linked to a synthetic meso-tetraarylporphyrin which successfully exhibited the photophysical functions of cartenoids in photosynthesis were first reported by G. Dicks, A. Moore, T. Moore and D. Gust in Photochemistry and Photobiology, Vol. 32, pp. 277-280 (Permagon Press Ltd. Great Britain, 1980).
A carotenoporphyrin which demonstrated quenching of the porphyrin triplet state by the attached carotenoid via triplet-triplet energy transfer was reported by R. V. Bensasson, E. J. Land, A. L. Moore, R L. Crouch, G. Dicks, T. A. Moore and D. Gust in Nature, Vol. 290, No. 5804, pp. 329-332 (Mar. 16, 1981). Since that time, various compounds which exhibit such triplet-triplet energy transfer have been reported. In 1984, five carotenoporphyrins were prepared by Dr. Paul Liddell at Arizona State University and reported in his doctoral thesis dated December 1985. Three carotenoporphyrins were reported by H. Frank, B. Chadwick, J. Oh, and D. Gust et al. in Biochemical et Biophysical Acta 892 (1987), pp. 253-263.
It has also been previously shown in U.S. Pat. No. 5,286,474, incorporated by reference herein, that certain synthetic carotenoporphyrins preferentially localize in mammalian pathology or target tissue where they absorb and emit light when irradiated with light so that the site of the pathology or target tissue may be detected by the fluorescence of the localized carotenoporphyrin.
This invention comprises the use of at least one auxiliary chromophore, such as carotenoids and other polyenes, to prevent undesired damage to tissue that can be caused by agents, as described above. The auxiliary chromophore is placed in the vicinity of the agent, through chemical bonding or other means, in such a way that it rapidly removes the excitation energy of the agent before that energy can cause substantial damage to the organism or sensitize the formation of singlet oxygen or other harmful species. The energy is removed through triplet-triplet energy transfer, single-singlet energy transfer, or other quenching phenomena. The energy thus acquired by the auxiliary chromophore is in a form that is essentially harmless to the organism, and is rapidly dissipated in a harmless way.
Localization of the imaging material employed in the practice of this invention is advantageous over the use of porphyrins alone. In addition, photodamage of tissue is advantageously precluded by the quenching of the porphyrin triplet state, and most importantly, photopenetration of the tissue by excitation light and by emitted light is enhanced by the use of agents that absorb and emit lower energy (longer wavelength) electromagnetic radiation. Thus this invention overcomes the problem of collateral tissue damage inherent with the use of existing photosensitizing compounds as diagnostic agents, and increases the detection of pathology or target tissues by using longer wavelength electromagnetic radiation.
This invention also provides a method of locating and visualizing mammalian pathology or target tissue. The method comprises administering a diagnostically effective amount of an imaging material, comprising an agent and at least one auxiliary chromophore, to a mammalian host, permitting the diagnostic agent to localize in the pathology or target tissue and thereafter irradiating the mammalian host with low energy electromagnetic radiation having a wavelength between about 600 and 1100 nm. The localized imaging material thus absorbs and in some cases fluoresces, or otherwise luminesces sharply defining the pathology or target tissue. Light absorbed by or emitted from the pathology or target tissue by the localized diagnostic agent employed in this invention sharply defines the location of the pathology or target tissue to be removed or otherwise treated. At the same time, the auxiliary chromophore prevents or limits photodamage to healthy tissue by quenching excited triplet states of the agent. In the event that the imaging process involves a process other than absorption or emission of light, the auxiliary chromophore still provides protection from photodamage by adventitious light in any tissues.
Generally, the imaging materials of the present invention may be effectively administered to representative mammals in dosages of from 0.5 to 50 xcexcmol/kg of host body weight, preferably from 3 to 48 hours prior to the diagnostic procedure or surgery.
The imaging materials employed in this invention have a number of advantages over current contrast agents and pathology or target tissue diagnostic procedures. They lack toxicity due to the antioxidant behavior of the auxiliary chromophore and the use of lower energy, higher wavelength electromagnetic radiation. Many individuals are sensitive to present X-ray contrast agents employed, for example in computer assisted tomography (CAT) scans. and there have been cases of severe allergy reactions resulting in anaphylactic shock. In addition, the patient is exposed to ionizing radiation. Alternatively, nuclear scans are often employed. However, nuclear scans require the administration of radioactive diagnostic materials and further, are useful primarily to define function as opposed to structure. Magnetic resonance imaging is accurate and definitive for the diagnosis of brain and other abnormalities but is expensive, unpleasantly noisy, confining for claustrophobic individuals, and often times the contrast agent enhancement is not target specific. This invention is advantageous over these alternative techniques. Lower cost is an additional advantage of this invention.
The present invention also provides an improved, more convenient and economical synthesis of the diagnostic agents employed herein.
An imaging material comprising at least two parts is employed in this invention. One part is an agent which localizes in pathology or target tissue in preference to surrounding tissue, absorbs light to produce an excited state, and allows imaging, demarcation, detection or diagnosis of the pathology or target tissue, or combinations thereof, via the absorption of light, or the emission of fluorescence, phosphorescence or heat. These effects can be detected via absorption spectroscopy, fluorescence or phosphorescence spectroscopy, or heat detection by calorimetric methods such as photothermal, photoacoustic, or mirage effect. Alternatively, the agent can enhance imaging as a magnetic resonance imaging contrast agent or by bearing radioactive isotopes of any of various elements whose radioactive emissions can be detected and used for imaging and diagnostic purposes, or by acting as a contrast agent for imaging by X-radiation or other high-energy radiation. The second part comprises an auxiliary chromophore, such as a carotenoid, polyene, or other chromophore with a low-energy triplet, singlet or charge-separated state, which is held in the vicinity of the agent through chemical bonding or other mechanisms, and which prevents damage to the organism by quenching high-energy states of the agent via triplet-triplet energy transfer, singlet-singlet energy transfer, electron transfer, or similar mechanisms.
Examples of preferred imaging materials include 
wherein
R1=hydrogen, alkyl or alkoxy groups; optionally the alkyl or alkoxy groups may include other groups such as COOH groups and the like,
R2=alkyl or aryl groups; the alkyl or aryl groups may include other groups such as COOH groups, NH2 groups and the like; 
wherein
R1=hydrogen, alkyl or alkoxy groups; the alkyl or alkoxy groups may include other groups such as COOH groups, NH2 groups and the like,
R2=hydrogen, alkyl or aryl groups; the alkyl or aryl groups may include other groups such as COOH groups, NH2 groups and the like; 
wherein
R1=hydrogen, alkyl or alkoxy groups, wherein at least one R1 is an alkoxy group; the alkyl or alkoxy groups may include other groups such as COOH groups and the like,
R2=hydrogen, alkyl or aryl groups; the alkyl or aryl groups may include other groups such as COOH groups, NH2 groups and the like; and 
wherein
R1=alkyl, alkoxy or aryl groups, the alkyl, alkoxy and aryl groups may include COOH groups and the like,
R2=alkyl or aryl groups, the alkyl and aryl groups may include COOH groups, NH2 groups and the like.
Administration of the imaging material to a human or other organism via methods including ingestion, injection (either alone or with a carrier such as an emulsifier, liposomes, or other biocompatible materials), inhalation or direct topical or internal application is followed by movement of the imaging material through the tissues of the organism, and concentration of the imaging material in pathological tissue, relative to surrounding tissue. The pathology or target tissue may be optically imaged, delineated, detected or diagnosed by: (i) illuminating the area containing the preferentially localized imaging material with light at any wavelength absorbed by the imaging material; and (ii) the absorption or the emission of light through fluorescence or phosphorescence or the generation of heat by this material. Detection of absorption or emission of light can be by the human eye, photomultiplier, photosensitive diode, or by any suitable device for detection of light at the appropriate wavelengths. Heat can be detected by photothermal, photoacoustic, mirage effect, or other calorimetric methods. Alternatively, if the agent is a magnetic resonance contrast agent, detection is by magnetic resonance imaging instrumentation. If the agent contains a radioactive isotope, radiation from this isotope can be detected. If the agent is an X-ray contrast material, pathology or target tissue can be imaged and identified using X-ray radiological techniques.
The optical agent, after excitation with light, will form excited singlet states, and may form excited triplet states or other high-energy states. In general, such states can react chemically with the organism, causing tissue damage, or react with oxygen to form harmful singlet oxygen or radical species, or with other molecules that may be present to produce other reactive, high energy species than may in turn react with the organism, causing tissue damage. In the imaging material, the auxiliary chromophore quenches the excited state or other high-energy state of the agent via an energy or electron transfer process. This quenching process produces a low-energy state, such as the polyene triplet state, or singlet state, which is incapable of harming the organism, and is incapable of sensitizing the formation of singlet oxygen or other reactive species. The method of coupling the auxiliary chromophore to the agent and the proper choice of agent and auxiliary chromophore ensure that this quenching is a rapid process. The method of coupling the auxiliary chromophore to the agent is facilitated through the overlap of electronic orbitals which may occur by either chemical bonding, Van der Waals interaction, or by using a coupling compound such as a protein to hold the auxiliary chromophore and the agent in near proximity. This rapid quenching deactivates the high energy triplet or other state of the agent before it can interact deleteriously with tissue, sensitize singlet oxygen formation, or react with other molecules that may be present to form a harmful species. In essence, the auxiliary chromophore allows the agent to perform as an imaging or detection agent, but prevents it from significantly damaging the organism.
In this invention, a method of administering an imaging material that will preferentially localize in pathology or target tissue and absorb and emit light without damaging the mammalian host upon irradiation with light comprises the steps of administering a diagnostically effective amount of the imaging materials and allowing said imaging material to circulate and accumulate and localize in pathology or target tissue, preferably from about 3 to 48 hours prior to the diagnostic or surgical procedure, and exposing the mammalian host to light causing the imaging material to absorb and fluoresce and thereby permitting visualization and definition of the pathology or target tissue to be removed or treated, or using some other visualization method such as those described hereinabove.
More specifically, the imaging material employed in the practice of this invention localizes in pathology or target tissue, absorbs light of one wavelength, may emit light of another wavelength, but does not damage healthy tissue. The process of this invention may be used both for diagnosis and as a valuable adjunct to surgery as light emitted from the pathology or target tissue by the localized agent in the imaging material would sharply define the location of the pathology or target tissue to be removed.
In practice, an imaging material is administered intravenously to a mammalian host in a dosage of from 0.5 to 50 xcexcmol/kg of body weight from 1 to 72 hours prior to exposure to radiation having a wavelength of from about 600 to about 1,100 nanometers. The imaging materials may be conveniently administered either solubilized in a biocompatible emulsion such as a Tween-80, CREMOPHOR EL emulsion (Sigma Chemical Company) or other suitable lipophilic emulsion or incorporated into liposomes such as unilamellar or multilamellar liposomes of a synthetic lipid such as dipalmitoylphosphatidylchotine (DPPC) sold by Sigma Chemical Company, Inc. After the photosensitive imaging material has had sufficient time to circulate and localize in pathology or target tissue, the mammalian host is exposed to a light so that the imaging material localized in pathology or target tissue absorbs and fluoresces permitting visualization of the pathology or target tissue location, size and configuration. The most suitable electromagnetic radiation sources, e.g. light sources, are those that emit radiation at wavelengths of between 600 to about 1,100 nanometers. The imaging agents of this invention have their red-most absorption bands in the range of about 600 to 1,100 nanometers. The use of imaging materials that absorb electromagnetic radiation in the 600-1,100 nm region permits the diagnostic light to penetrate the tissue more deeply and image deeper pathology or target tissue tissues than can be obtained with molecules that absorb only at shorter wavelengths. Thus, monochromatic radiation at these wavelengths would be preferentially absorbed. In addition to using the human eye as a detector, a light-sensitive electronic device such as a photomultiplier or photodiode array could be used as a detector to provide a picture or electronic image of localized material. Alternatively, any other detection and visualization method for the agent may be employed. Even if light is not part of the diagnostic process, the auxiliary chromophore will protect the skin and other tissues of the organism from photodamage by adventitious light absorbed by the agent.